Production and characterization of lipopeptide biosurfactant from a new strain of Pseudomonas antarctica 28E using crude glycerol as a carbon source

Pseudomonas is a cosmopolitan genus of bacteria found in soil, water, organic matter, plants and animals and known for the production of glycolipid and lipopeptide biosurfactants. In this study bacteria (laboratory collection number 28E) isolated from soil collected in Spitsbergen were used for biosurfactant production. 16S rRNA sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) revealed that this isolate belongs to the species Pseudomonas antarctica. In the present study, crude glycerol, a raw material obtained from several industrial processes, was evaluated as a potential low-cost carbon source to reduce the costs of lipopeptide production. Among several tested glycerols, a waste product of stearin production, rich in nitrogen, iron and calcium, ensured optimal conditions for bacterial growth. Biosurfactant production was evidenced by a reduction of surface tension (ST) and an increase in the emulsification index (E24%). According to Fourier-transform infrared spectroscopy (FTIR) and electrospray ionization mass spectrometry (ESI-MS), the biosurfactant was identified as viscosin. The critical micelle concentration (CMC) of lipopeptide was determined to be 20 mg L−1. Interestingly, viscosin production has been reported previously for Pseudomonas viscosa, Pseudomonas fluorescens and Pseudomonas libanensis. To the best of our knowledge, this is the first report on viscosin production by a P. antarctica 28E. The results indicated the potential of crude glycerol as a low-cost substrate to produce a lipopeptide biosurfactant with promising tensioactive and emulsifying properties.


Introduction
Surfactants are described as active ingredients, found in household and industrial-scale cleaning agents. 1 Their activity derives from the surface or interfacial tension reduction properties, which increase the solubility of non-aqueous phase liquids. Despite their numerous benets, the high toxicity of surfactants is not without signicance. 2 The negative environmental impact is related to the production process, occurring via chemical synthesis. Moreover, surfactants' recalcitrant and persistent nature entails a slow rate of degradation. Despite high environmental toxicity, chemically derived surfactants are still in wide scale application. 3 However, in a time of ecological crisis there is a growing demand for environmentally friendly agents. 1 Therefore, biosurfactants are increasingly taking the place of their synthetic counterparts. Biosurfactants are surfaceactive compounds physiologically secreted by microorganisms for utilization of hydrocarbons. 4 Microbial surfactants exhibit superior properties compared to chemically manufactured alternatives, of which the most important is outstanding surface activity, stability in a wide range of pH and temperature, biodegradability, low toxicity and extraordinary emulsifying and demulsifying activity. 5 Numerous genera have been described for biosurfactant production, with Bacillus spp., Acinetobacter spp., Rhodococcus spp., Halomonas spp. and Candida spp. being the best-known biosurfactant producers, as well as Pseudomonas, described as a dominant genus involved in biosurfactant synthesis. 6 Pseudomonas is a cosmopolitan microorganism found in miscellaneous habitats including soil, water, organic matter, plants and animals. 7 Due to its metabolic complexity, Pseudomonas is

Materials and methods
Identication studies P. antarctica strain 28E, previously isolated from soil samples collected along western and central parts of Spitsbergen, Svalbard Archipelago 22 was identied by the analysis of the 16S rRNA gene. To achieve better strain identication, matrixassisted laser desorption/ionization time-of-ight mass spectrometry (MALDI-TOF) was also employed. For sequencing, genomic bacterial DNA was isolated using the Bacterial & Yeast Genomic DNA Purication Kit (EURx, Poland). The concentration of the isolated DNA sample was determined using Nanodrop (WPA Biowave II, USA). PCR of isolated DNA was performed in a total value of 20 mL of reaction mixture, containing 10 mL of PCR MasterMix 2X (Thermo Scientic, USA), 1 mL of 27F (5 ′ -AGAGTTTGATCCTGGCTCAG-3 ′ ) and 1492R (5 ′ -GGTTACCTTGTTACGACTT3 ′ ) primers, genomic DNA, applied in a concentration of 50-100 ng, and nuclease-free water. PCR MasterMix 2X contained Taq DNA polymerase (0.05 U mL −1 ), reaction buffer, MgCl 2 (4 mM), and 0.4 mM of each dNTP. Reaction of polymerase was initiated by a pre-denaturation step performed at 95°C for 5 min, followed by 30 cycles of denaturation (95°C, 20 s), annealing (55°C, 30 s) and elongation (72°C, 2 min). The reaction was terminated with an extended elongation (72°C, 5 min). The PCR products were subjected to agarose gel electrophoresis, puried using a Gel-Out Kit for DNA extraction (A&A Biotechnology, Poland), and their nucleotide sequence was determined by the Genomed S.A. sequencing service (Warsaw, Poland).
Identication of P. antarctica 28E by the MALDI TOF MS method was carried out in the Microbiological Laboratory in the Jagiellonian Center of Innovation in Cracow, using a MALDI-TOF Microex LT mass spectrometer by Bruker (Bruker, USA). Sequences of 16S rRNA of P. antarctica 28E and results of MALDI-TOF study were then analyzed with the Basic Local Alignment Search Tool (BLAST) program, available on the National Center for Biotechnology Information (NCBI) website.

Media and growth conditions
The bacterial strain P. antarctica 28E was stored at the Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences (Wrocław, Poland) in the form of glycerol stock (20% v/v) at −80°C and, when needed, cultured in Luria-Bertani broth (LB; 10 g L −1 of tryptone, 5 g L −1 of yeast extract, and 10 g L −1 of NaCl; A&A Biotechnology, Poland) for 20 h with orbital agitation (180 rpm) at 28°C.
In addition, another glycerol-derived waste, previously characterized by Signori et al., 24 originating from industrial biodiesel production from palm oil (Milan (G7)) was included in this research. In addition, to estimate the suitability of crude glycerol as a potential substrate for biosurfactant synthesis, a process was additionally conducted using pure glycerol (G6) (99.5%) (Centro-Chem, Poland) as a control. The purity of crude glycerols was estimated using high-performance liquid chromatography (HPLC, UltiMate 3000, Dionex-Thermo Fisher Scientic, London, UK) equipped with a HyperRez Carbohydrate H + Column (Thermo Fisher Scientic, London, UK) and a refractive index (RI) detector (Shodex, Ogimachi, Japan). Tri-uoroacetic acetic acid at the concentration 25 mM was applied as an elution agent. Elution was carried out with a ow rate of 0.6 mL min −1 at 65°C. 25

Microplate cultures
Glycerols were sterilized and added in a proper ratio to the sterile MSM. Microplate cultures were conducted in The Spark multimode microplate reader (Tecan Group Ltd) for 120 h at 28°C with continuous orbital shaking (180 rpm). Cultures were carried out in a 96-well plate. Each well was lled with 200 mL of the medium, inoculated with 1% LB bacterial culture, performed as described in section media and growth conditions. Optical density (OD 600 ) was measured each hour in 120 cycles. All measurements were carried out in triplicate.

Studies on biosurfactant production
According to the results of microplate cultures, further investigations were performed using G5 glycerol, ensuring the best growth of P. antarctica 28E. The biosurfactant production was carried out in 250 mL baffled Erlenmeyer asks containing 50 mL of MSM medium, supplemented with 2% and 4% waste glycerol. Cultures were inoculated with 1% 20 h LB bacterial inoculum and incubated at 28°C for 96 h with orbital agitation (180 rpm). As a control, uninoculated medium, treated with the same conditions, was also monitored. OD 600 , pH, glycerol utilization, surface tension (ST) and emulsication index (E 24 %) were determined with 24 h intervals. OD 600 was assessed over time for the culture medium against a control medium. To determine the pH, glycerol utilization, ST and E 24 % of the culture medium, one ask was collected each day, centrifuged for 10 min at 7500 rpm and ltered through 0.22 mm membrane for biomass separation. Glycerol utilization was examined using HPLC by comparing glycerol concentration in the culture supernatant to the standard of a known concentration. 25 Surface tension was determined at 25°C using Krüss Force Tensiometer K6 (Krüss, Germany), calibrated with ultra-pure water according to du Nouy's ring method. 26 To determine E 24 %, 2 ml of cell-free supernatant was mixed with 2 mL of nhexadecane in a glass tube and vortexed for 2 min. Then, the mixture was le to stand for 24 h and the height of the liquid and emulsion layer was measured. 27 E 24 % was calculated according to eqn (1), as follows: height of the emulsion layer total height of the mixtures Â 100 (1)

Preparation of crude biosurfactant solution for characterization studies
To produce biosurfactant in a large-scale system composed of three 1000 mL baffled Erlenmeyer asks containing 200 mL of MSM supplemented with 2% (w/v) of G5 glycerol, inoculum was applied in a concentration of 1% (v/v). Culture was conducted at 28°C with rotary agitation of 180 rpm for 72 h. Then, the postculture medium was centrifuged at 7500 rpm for 10 min and ltered through 0.22 mm membrane for biomass separation. The supernatant was lyophilized and resuspended in 20% (v/v) acetonitrile solution to obtain a biosurfactant concentration of approximately 0.17 g mL −1 . The biosurfactant solution was puried using a solid phase extraction (SPE) system, equipped with cartridges of the Chromabond C18 SPE system (Macherey-Nagel, Germany) and eluted using an acetonitrile gradient (0%, 20%, 50%, 80% and 100% acetonitrile-water (v/v) solution). Each elution step was followed by HPLC (Shimadzu, Kyoto, Japan) performed according to the methodology described by Ciurko et al. 28 to determine the composition of the solvent ensuring the most efficient biosurfactant elution. Therefore, 100 mL of the sample was dissolved in 900 mL of methanol (Chempur, PiekaryŚląskie, Poland). As the mobile phase, solvents A (0.1% triuoroacetic acid) and B (0.1% triuoroacetic acid in acetonitrile) were applied in the following order: ( Samples were injected in a 10 mL volume on a Hypersil GOLD column (5 mm; 4.6 × 150 mm) and eluted for 25 min. Elution was performed with a ow rate of 0.5 mL min −1 and detection was conducted at 210 nm wavelength. Each determination was performed in triplicate.

Analysis of the biosurfactant chemical structure
Fourier-transform infrared spectroscopy (FTIR). The structure of the puried biosurfactant was examined using FTIR and compared to the standard of surfactin ($98.0%) (Sigma-Aldrich, Germany) and rhamnolipid (>90%) (Sigma-Aldrich, Germany). The IR spectra were observed using the IRSpirit FTIR spectrometer (Shimadzu, Kyoto, Japan) at room temperature (25°C). The main functional groups were observed between 400 and 4000 wavenumbers (cm −1 ) at a resolution of 2 cm −1 .
Electrospray ionization mass spectrometry (ESI-MS). The biosurfactant sample was identied by electrospray ionization (ESI) mass spectrometry (MS) using a Compact Mass Spectrometer (Bruker Daltonics, Bremen, Germany) in the positive ionization mode, using the following settings: capillary voltage 3500 V, nebulizer 1.5 bar, dry gas 8 L min −1 , dry temperature 180°C. Data were collected for 50-3000 m/z. Next, the data obtained were processed with the Compass DataAnalysis 4.2 soware package (Bruker, Bremen, Germany).
Determination of physicochemical properties of viscosin. According to the result of HPLC analysis, 50% (v/v) acetonitrile solution, containing the biosurfactant, was evaporated and lyophilized to remove acetonitrile impurities. Then, the ne powder of biosurfactant was examined to determine the concentration, ensuring formation of micelles. Therefore, a series of biosurfactant dilutions in a concentration range of 0-250 mg L −1 was analyzed in terms of ST. 26 A KRÜSS K6 Tensiometer (KRÜSS GmbH, Germany) equipped with a 1.9 cm De Noüy platinum ring was used at room temperature (25°C). The CMC was determined graphically by plotting the relationship between ST and biosurfactant concentration. All the measurements were made in triplicate.
In order to verify the wetting ability of the viscosin solutions, contact angle measurements were carried out using the OCA 15EC system (DataPhysics, Filderstadt, Germany), and the drop shape was analyzed with SCA20 soware (DataPhysics). For each measurement, a sample droplet of 0.2 mL was placed on a solid support. Silicone, polystyrene and glass surfaces were used as supports. All measurements were made at ambient temperature (25°C) in a saturated vapor atmosphere to reduce droplet evaporation. The contact angle value is reported as the average of ten measurements.

Identication of bacterium
The sequence of 16S rRNA of the isolated bacterium showed 95.47% identity to P. antarctica CMS 35 (DSM 15318). The same score was obtained for several species within the genus Pseudomonas. However, results of MALDI-TOF clearly indicated species affiliation, with the identication rate of 2.13. Therefore, the isolated bacterium was affiliated to P. antarctica.
The impact of post-production glycerols on P. antarctica 28E growth curves Crude glycerols were identied as a proper substrate for the growth of P. antarctica 28E, favoring an increased growth rate. The origin of crude glycerol was as follows: G1 derived from soap production, G2-G4 derived from biodiesel production, and G5 derived from stearin production. Pure glycerol (G6) was used as a reference carbon source ( Table 1).
The OD 600 of bacterial cells in MSM medium, supplemented with G5 glycerol, was the highest among all tested variants (Fig. 1). Inversely, in the culture growing on pure glycerol, the number of bacterial cells was the most limited. This has been noted for both the 2% (w/v) (Fig. 1A) and 4% (w/v) (Fig. 1B) glycerol cultures. This should be explained by the lack of nutrients essential for bacterial growth in the reagent glycerol composition. Therefore, decits of nitrogen and trace elements showed a negative effect for culture development. In the cultures performed with G7 glycerol addition, the P. antarctica 28E growth curves presented an irregular shape, suggesting the inhibitory effect of the substrate on the microbial community. We did not observe a signicant increase in bacteria concentration; therefore, G7 glycerol was not included in the curves of bacterial growth (data not published) and was not considered for the future stage of studies.
The composition of G5 glycerol, recognized as the best substrate for P. antarctica 28E growth, was distinguished by the highest nitrogen content (Table 1). In addition, G5 glycerol was characterized by high content of iron and calcium. While the positive effect of nitrogen on microbial metabolism is well known, participating in the synthesis of amino acids, DNA, RNA and ATP among other molecules, the impact of iron and calcium is not so well explored. 29 The high concentration of iron and calcium in G5 glycerol signicantly supported the growth of P. antarctica 28E. Calcium, in prokaryotes, is involved in a several metabolic processes such as maintenance of cell structure, motility, cell division, gene expression and cell differentiation processes such as sporulation, heterocyst formation and fruiting body development. Therefore it is essential for bacterial colony evolution. 30 However, the direct effect of calcium on the growth of Pseudomonas bacteria has not been documented so far. Therefore, this is a rst report showing benecial effect of calcium on Pseudomonas bacteria growth. On the other hand, several studies have highlighted the positive effect of iron, observed in our research in relation to P. antarctica 28E, on the growth of the genus Pseudomonas. Iron, applied in three different forms, i.e. FeSO 4 , Fe(NO 3 ) 3 and FeCl 3 , at the concentration of 0.1 mM, signicantly supports the growth and biosurfactant production of Pseudomonas citronellolis 620C. 31 Moreover, among the tested iron sources, the FeCl 3 supported the microbial growth to the greatest extent. The explanation is the highest solubility of FeCl 3 in comparison to Fe(NO 3 ) 3 and FeSO 4 . Other studies evaluated the effect of different forms and concentrations of iron on the growth rate of three biosurfactantproducing species (P. citronellolis (isolate 22A), P. aeruginosa (isolate 312A) and P. aeruginosa (isolate 332C)) conrmed observed in our studies promoting effect of iron on Pseudomonas growth. 32 Several iron forms (FeCl 3 , Fe(NO 3 ) 3 , Fe-EDTA, FeSO 4 , Fe 0 ) at the concentration of 0.1 mM showed a positive effect on bacterial growth, increasing the cell number. The exception was iron oxide (Fe 2 O 3 ), of limited solubility, which did not differ from the control. 32 Finally, Kim et al. 33 searched for a nutrient affecting the growth of Pseudomonas syringae pv. tomato DC3000. Aer a series of experiments, iron supplementation was found to induce a strong positive response for bacterial growth what is in line with our studies. Bacteria presented a dose-dependent growth enhancement. The addition of 50 mM of Fe 3+ caused an OD 600 change of 0.35, while supplementation with 100 mM resulted in an increase of 0.74. Based on the research described above and on the result presented in this study, the crucial role of iron on the growth of Pseudomonas bacteria must be emphasized. The same as the concentration applied, the bioavailability for the microbial community is of decisive importance. The post-production glycerols applied in this research, in particular G5 glycerol, meet the nutritional requirements of P. antarctica 28E, providing appropriate conditions for bacterial growth.

Kinetics of biosurfactant production
Based on the results described above, G5 glycerol was applied as a substrate for biosurfactant production. In the ask culture, containing either 2% (w/v) or 4% (w/v) of the substrate, P. antarctica 28E was growing properly, as indicated by high OD 600 (Fig. 2). The plateau was reached aer 72 h of cultivation, for both 2% (w/v) (OD 600 13.01 ± 0.4) and 4% (w/v) (OD 600 11.28 ± 1.15) glycerol concentration. Interestingly, the OD 600 of bacterial cells was higher in 2% (w/v) glycerol medium. The  explanation should be sought for in the high concentration of iron in G5 glycerol. In the 4% (w/v) glycerol medium, aer 72 h of growth, the culture entered the death phase, despite the fact that almost 50% of the substrate was le (Fig. 2). In the 2% (w/v) glycerol medium the substrate was utilized within 72 h, leading to the death of the culture. According to Tsipa et al. 31 the borderline between iron deciency and toxicity is narrow. Iron is a key component of cytochromes and contributes to the Krebs cycle. In addition, it participates in the protection of the cells against superoxide radicals. However, if the iron concentration in the environment exceeds the limit, it can interact with reactive oxygen species, forming greatly damaging hydroxyl radicals. In our study, 2% (w/v) glycerol supplementation was below the nutritional requirements of P. antarctica 28E, while 4% (w/v) probably exceeded the limit of iron toxicity. Therefore, to ensure appropriate development of P. antarctica 28E culture, optimization studies should be performed. However, this is an issue for another study, while the aim of this work was to investigate the capacity of P. antarctica 28E to produce biosurfactant using waste substrates.
According to Moshtagh et al. 34 the value of pH can affect microbial growth and reproduction signicantly inuencing the absorption of nutrients and activity of the enzymes engaged in several metabolic processes. In our studies, the pH of P. antarctica 28E culture growing in 2% (w/v) and 4% (w/ v) concentration of glycerol was at a stable value of around 8 (data not shown) and it was applicable for the growth and biosurfactant synthesis of bacteria. However, numerous studies have shown that the optimal pH for biosurfactant production by Pseudomonas bacteria seems to be a species-or strain-dependent feature. As in our studies, the culture of Pseudomonas aeruginosa RS29 pH in a range of 7-8 ensured the most efficient biosurfactant production in the glycerolbased medium. 35,36 Surprisingly, the production of biosurfactant in Pseudomonas aeruginosa TMN culture was the most efficient at pH 7 and a slight alkalization to pH 8 caused a dramatic change in the yield of biosurfactant production, reducing it greatly. 37 Production of biosurfactant by P. antarctica 28E was indicated by the reduction of the surface tension (ST), both in 2% (w/v) and 4% (w/v) glycerol medium (Fig. 2). Signicant ST reduction was observed in the early exponential phase of growth, indicating that P. antarctica 28E biosurfactant consists of primary metabolites, synthesized simultaneously with the formation of cellular biomass. Similar observations have been made while studying P. aeruginosa biosurfactant production. 38 In P. antarctica 28E culture, a signicant drop of the ST, from the initial value of 59 mN m −1 to 28 mN m −1 (Fig. 2A) and from 52 mN m −1 to 27 mN m −1 (Fig. 2B), respectively, in the 2% (w/v) and 4% (w/v) glycerol medium was observed. However, in the culture supplemented with 4% (w/v) glycerol, ST dropped signicantly during 24 h and reached a stable value of approximately 27 mN m −1 , while in the 2% (w/v) glycerol medium, aer ST decline, a slight increase was observed. It indicated reduction of the biosurfactant concentration in the 2% (w/v) glycerol medium. The ST increase occurred aer glycerol stores were depleted. Therefore, to provide the bacteria with nutrients, the already produced biosurfactants could possibly be hydrolyzed.
A variety of microorganisms of the genus Pseudomonas produce biosurfactants that are diverse in chemical composition. The nature and amount of the biosurfactant produced depends on the species of Pseudomonas and the various nutritional factors available for their growth ( Table 2).
Glycerol is a frequently chosen substrate in the research on biosurfactant production in the Pseudomonas genus, while most of these studies concern rhamnolipid synthesis. 35,36 Biodiesel side stream waste glycerol was applied for biosurfactant production using Pseudomonas aeruginosa RS6. At optimal fermentation conditions, ST declined from 72.13 mN m −1 to 29.4-30.4 mN m −1 ; therefore biosurfactant activity was reported to be better than that of some chemical-based surfactants and similar to the activity observed in our studies. 49 Corresponding to our research ST drop was observed in another study, where glycerol was applied as a substrate for anaerobic production of rhamnolipids. Three strains of P. aeruginosa (SG, L6-1 and FA1) reduced the ST at different substrate concentrations from 63 Biosurfactant production in the P. antarctica 28E culture affected emulsifying properties of the cultivation medium was indicated in addition by the growing E 24 %. In the medium supplemented with 2% as well as 4% glycerol, emulsication index (E 24 %) reached a maximum value of 33% (data not shown). However, obtained result was lower than that observed in the research of da Rosa et al. 53 where in the optimal conditions for rhamnolipid production E 24 % reached the value of 61%. In another study, where the halophilic bacterium Pseudomonas stutzeri BK-AB12 was cultivated in medium containing 3% glycerol, an increase of E 24 % reach the higher value of 53.33%. Finally, the cell-free supernatant of P. aeruginosa JBK1 culture, cultivated in 3% biodiesel derived waste glycerol medium, exhibited stable emulsifying properties with all the hydrocarbons tested. The best E 24 % results; 62% obtained in relation to kerosene and xylene and 60% obtained using hexadecane 38 were almost twice as high as in our research. Against the background of the presented results, the E 24 % of P. antarctica 28E culture supernatant is relatively low. According to Jain et al. 54 high molecular weight biosurfactant acts as an emulsion stabilizer, while low molecular mass compounds are unable to create stable emulsions. This indicates the presence of the low molecular weight biosurfactant in P. antarctica 28E culture medium.

Report on biosurfactant characterization studies
Lipopeptide nature of P. antarctica 28E biosurfactant indicated by a Fourier-transform infrared spectroscopy (FTIR). FTIR was used in order to verify the chemical nature of P. antarctica 28E biosurfactant. We observed the characteristic peaks at 3286 cm −1 and 1646 cm −1 corresponding to the N-H and CO-N stretching vibrations respectively (Fig. 3A). In addition, absorption bands at 2954 cm −1 , 2926 cm −1 and 2869 cm −1 assigned to asymmetric C-H stretching vibrations were detected. The presence of aliphatic chains (-CH 3 , -CH 2 -) was conrmed by peaks at 1463 cm −1 and 1394 cm −1 . Another peak, detected at 1063 cm −1 indicated the presence of C-O bonds in the biosurfactant structure. Summing up, the observed peaks displayed the presence of aliphatic hydrocarbons attached to a peptide moiety. Therefore the biosurfactant of P. antarctica 28E was classied as a lipopeptide. Corresponding peaks were observed on the surfactin standard spectrum (Fig. 3B). Moreover, Liu et al., 55 Thavasi et al. 56 and Pardhi et al. 57 detected similar absorption peaks while studying lipopeptides secreted by P. aeruginosa SNP0614, P. aeruginosa and Pseudomonas guguanensis D30, respectively.
Structural characterization of P. antarctica 28E lipopeptide.   61 Therefore, lipopeptide biosurfactant obtained in our studies were identied as viscosin.
As the nal stage of this study the CMC of viscosin was determined. The CMC reached 20 mg L −1 (Fig. 5), which is in line with other results, i.e. 15 mg L −1 , 50 mg L −1 and 54 mg L −1 , presented in the work of Hamley, 62 Portet-Koltalo et al. 63 and Saini et al., 21 respectively.
The viscosin biosurfactant was studied for any effect on changes in the contact angle on hydrophobic and hydrophilic surfaces. Silicone and polystyrene surfaces, which have high hydrophobicity, are used regularly in biomedical industries. The largest reduction in contact angle was observed on silicone (from 92°to 77°) and polystyrene (from 75°to 61°) at 0.02% (w/ v) viscosin (Table 3).
However, on a highly hydrophilic surface like glass, there was no signicant reduction in contact angle values (from 6°to 5°) for 0.02% (w/v) viscosin solution (Table 3). For comparison, Al-Wahaibi et al. 64 reported changes in the wettability of a hydrophobic surface from 59°to 29°by biosurfactant produced by B. subtilis B30. Al-Sulaimani et al. 65 also reported that biosurfactant produced by B. subtilis W19 changed the contact angle of distilled water from 70.6°to 25.3°at 0.25% (w/ v) biosurfactant. In conclusion, our results suggest that the  viscosin changed the wettability of hydrophobic surface toward more water-wet, which is benecial during enhanced oil recovery (EOR) applications.

Conclusion
Selection of suitable substrates for lipopeptide production by Pseudomonas species is of great importance. The proper substrate should be inexpensive and renewable, and provide adequate conditions for bacterial growth. Our work has shown that glycerol, as the sole carbon and energy source, has great potential for increasing cell growth. Signicant impacts of iron, calcium and nitrogen ions on P. antarctica 28E culture growth and biosurfactant production were observed. Importantly, iron concentration was found to stimulate or inhibit Pseudomonas growth, suggesting the importance of its adjustment in the medium. The pH value was also found to signicantly affect the biosurfactant production. Furthermore, P. antarctica 28E is able to produce a lipopeptide viscosin (1125 Da). Moreover, pH 8 is suitable for lipopeptide production. Viscosin demonstrated signicant activity, reducing ST considerably in 2% (w/v) and 4% (w/v) glycerol medium. In addition, high viscosin activity was manifested by the low CMC, determined to be 20 mg L −1 , which is in line with previously published data. The low CMC as well as the production using cheap, waste substrates supports the economic advantages for possible future large-scale viscosin application.
This study provides the rst evidence that viscosin can be produced using P. antarctica 28E in a culture medium containing crude glycerol. To reduce the costs of biosurfactant production, stearin-derived glycerol was used as a promising cheap and renewable carbon source. The properties of the biosurfactant obtained have potential application as a cleaning and emulsifying agent in the pharmaceutical and food industry and/or bioremediation of hydrocarbon-contaminated sites.